Long-distance mobile mRNA CAX3 modulates iron uptake and zinc compartmentalization.

Iron deficiency in plants can lead to excessive absorption of zinc; however, important details of this mechanism have yet to be elucidated. Here, we report that MdCAX3 mRNA is transported from the leaf to the root, and that MdCAX3 is then activated by MdCXIP1. Suppression of MdCAX3 expression leads to an increase in the root apoplastic pH, which is associated with the iron deficiency response. Notably, overexpression of MdCAX3 does not affect the apoplastic pH in a MdCXIP1 loss-of-function Malus baccata (Mb) mutant that has a deletion in the MdCXIP1 promoter. This deletion in Mb weakens MdCXIP1 expression. Co-expression of MdCAX3 and MdCXIP1 in Mb causes a decrease in the root apoplastic pH. Furthermore, suppressing MdCAX3 in Malus significantly reduces zinc vacuole compartmentalization. We also show that MdCAX3 activated by MdCXIP1 is not only involved in iron uptake, but also in regulating zinc detoxification by compartmentalizing zinc in vacuoles to avoid iron starvation-induced zinc toxicity. Thus, mobile MdCAX3 mRNA is involved in the regulation of iron and zinc homeostasis in response to iron starvation.

The vacuolar Ca2+ transporter CATION EXCHANGER 2 regulates cytosolic calcium homeostasis, hypoxic signaling, and response to flooding in Arabidopsis thaliana.

Flooding represents a major threat to global agricultural productivity and food security, but plants are capable of deploying a suite of adaptive responses that can lead to short- or longer-term survival to this stress. One cellular pathway thought to help coordinate these responses is via flooding-triggered Ca2+ signaling. We have mined publicly available transcriptomic data from Arabidopsis subjected to flooding or low oxygen stress to identify rapidly upregulated, Ca2+ -related transcripts. We then focused on transporters likely to modulate Ca2+ signals. Candidates emerging from this analysis included AUTOINHIBITED Ca2+ ATPASE 1 and CATION EXCHANGER 2. We therefore assayed mutants in these genes for flooding sensitivity at levels from growth to patterns of gene expression and the kinetics of flooding-related Ca2+ changes. Knockout mutants in CAX2 especially showed enhanced survival to soil waterlogging coupled with suppressed induction of many marker genes for hypoxic response and constitutive activation of others. CAX2 mutants also generated larger and more sustained Ca2+ signals in response to both flooding and hypoxic challenges. CAX2 is a Ca2+ transporter located on the tonoplast, and so these results are consistent with an important role for vacuolar Ca2+ transport in the signaling systems that trigger flooding response.

Enhanced LC-ESI-MS/MS Sensitivity by Cationic Derivatization of Organophosphorus Acids.

The chemical derivatization to enhance the signal intensity and signal-to-noise (S/N) of several organophosphorus (OP) acids in liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS) is illustrated. The OP class of compounds represents the environmental degradants of OP nerve agents and pesticides. N-(2-(bromomethyl)benzyl)-N,N-diethylethanaminium bromide (CAX-B) was utilized to derivatize a panel of eight acids consisting of five alkyl methylphosphonic acids (ethyl-, isopropyl-, isobutyl-, cyclohexyl-, and pinacolyl-methylphosphonic acid) along with three dialkylphosphate analogs (diethyl-, dibutyl-, and diethyl thio-phosphate). The derivatization reaction with CAX-B was conducted in acetonitrile in the presence of potassium carbonate at 70 °C for 1 h. The resulting acid derivatives were analyzed with an LC-Orbitrap-ESI-MS/MS, and their dissociation processes were investigated. It was found that the derivatization procedure increased the limits of identification (LOIs) by one to over two orders of magnitude from the range of 1 to 10 ng/mL for the intact OP-acids to the range of 0.02-0.2 ng/mL for the derivatized acids utilizing an LC-MS(QqQ) in MRM mode, regardless of the sample matrix (hair, concrete, or plant extracts). The interpretation of the corresponding ESI-MS/MS spectra for each type of derivatized sub-OP family revealed the formation of characteristic neutral losses and a characteristic ion for the organophosphorus core. This derivatization is beneficial and useful for screening and identifying target and "unknown" OP acids.

Structure-Based Function and Regulation of NCX Variants: Updates and Challenges.

The plasma-membrane homeostasis Na+/Ca2+ exchangers (NCXs) mediate Ca2+ extrusion/entry to dynamically shape Ca2+ signaling/in biological systems ranging from bacteria to humans. The NCX gene orthologs, isoforms, and their splice variants are expressed in a tissue-specific manner and exhibit nearly 104-fold differences in the transport rates and regulatory specificities to match the cell-specific requirements. Selective pharmacological targeting of NCX variants could benefit many clinical applications, although this intervention remains challenging, mainly because a full-size structure of eukaryotic NCX is unavailable. The crystal structure of the archaeal NCX_Mj, in conjunction with biophysical, computational, and functional analyses, provided a breakthrough in resolving the ion transport mechanisms. However, NCX_Mj (whose size is nearly three times smaller than that of mammalian NCXs) cannot serve as a structure-dynamic model for imitating high transport rates and regulatory modules possessed by eukaryotic NCXs. The crystal structures of isolated regulatory domains (obtained from eukaryotic NCXs) and their biophysical analyses by SAXS, NMR, FRET, and HDX-MS approaches revealed structure-based variances of regulatory modules. Despite these achievements, it remains unclear how multi-domain interactions can decode and integrate diverse allosteric signals, thereby yielding distinct regulatory outcomes in a given ortholog/isoform/splice variant. This article summarizes the relevant issues from the perspective of future developments.

Teaching CAD/CAM/CAE tools with project-based learning in virtual distance education.

Computer-aided design, manufacturing and engineering technologies (CAD/CAM/CAE) are a mainstay in today's industry and therefore they should be an important part in the current training plan of the graduate engineers. However, their implementation in the university environment presents certain barriers that make it difficult. In this work, we study the feasibility of the teaching proposal of the management of this type of tools through a Project-Based Learning method in a distance learning environment. The methodology has been implemented transversally in two Master's degree subjects related to advanced design and manufacturing and has been carried out thanks to the operation of the product lifecycle management platform software by virtual machines. The practice has given very good pedagogical results in the work of skills related to the field of industrial design and manufacturing. The virtual system has demonstrated high efficiency and students have shown a satisfactory evolution in their professional training.

CAX3 (cation/proton exchanger) mediates a Cd tolerance by decreasing ROS through Ca elevation in Arabidopsis.

KEY MESSAGE: Over-expression of CAX3 encoding a cation/proton exchanger enhances Cd tolerance by decreasing ROS (Reactive Oxygen Species) through activating anti-oxidative enzymes via elevation of Ca level in Arabidopsis CAXs (cation/proton exchangers) are involved in the sequestration of cations such as Mn, Li, and Cd, as well as Ca, from cytosol into the vacuole using proton gradients. In addition, it has been reported that CAX1, 2 and 4 are involved in Cd tolerance. Interestingly, it has been reported that CAX3 expressions were enhanced by Cd in Cd-tolerant transgenic plants expressing Hb1 (hemoglobin 1) or UBC1 (Ub-conjugating enzyme 1). Therefore, to investigate whether CAX3 plays a role in increasing Cd tolerance, CAX3 of Arabidopsis and tobacco were over-expressed in Arabidopsis thaliana. Compared to control plants, both transgenic plants displayed an increase in Cd tolerance, no change in Cd accumulation, and enhanced Ca levels. In support of these, AtCAX3-Arabidopsis showed no change in expressions of Cd transporters, but reduced expressions of Ca exporters and lower rate of Ca efflux. By contrast, atcax3 knockout Arabidopsis exhibited a reduced Cd tolerance, while the Cd level was not altered. The expression of Δ90-AtCAX3 (deletion of autoinhibitory domain) increased Cd and Ca tolerance in yeast, while AtCAX3 expression did not. Interestingly, less accumulation of ROS (H2O2 and O2-) was observed in CAX3-expressing transgenic plants and was accompanied with higher antioxidant enzyme activities (SOD, CAT, GR). Taken together, CAX3 over-expression may enhance Cd tolerance by decreasing Cd-induced ROS production by activating antioxidant enzymes and by intervening the positive feedback circuit between ROS generation and Cd-induced spikes of cytoplasmic Ca.

Combined analyses of translatome and transcriptome in Arabidopsis reveal new players responding to magnesium deficiency.

Translational control of gene expression, including recruitment of ribosomes to messenger RNA (mRNA), is particularly important during the response to stress. Purification of ribosome-associated mRNAs using translating ribosome affinity purification (TRAP) followed by RNA-sequencing facilitates the study of mRNAs undergoing active transcription and better proxies the translatome, or protein response, to stimuli. To identify plant responses to Magnesium (Mg) deficiency at the translational level, we combined transcriptome and translatome analyses. Excitingly, we found 26 previously unreported Mg-responsive genes that were only regulated at the translational level and not the transcriptional level, during the early response to Mg deficiency. In addition, mutants of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), the H+ /CATION EXCHANGER 1 and 3 (CAX1 and CAX3), and UBIQUITIN 11 (UBQ11) exhibited early chlorosis phenotype under Mg deficiency, supporting their functional involvement in ion homeostasis. Overall, our study strongly supports that TRAP-seq combined with RNA-seq followed by phenotype screening could facilitate the identification of novel players during stress responses.

The grapevine CAX-interacting protein VvCXIP4 is exported from the nucleus to activate the tonoplast Ca2+/H+ exchanger VvCAX3.

MAIN CONCLUSION: The nuclear-localized CAX-interacting protein VvCXIP4 is exported to the cytosol after a Ca2+ pulse, to activate the tonoplast-localized Ca2+/H+ exchanger VvCAX3. Vacuolar cation/H+ exchangers (CAXs) have long been recognized as 'housekeeping' components in cellular Ca2+ and trace metal homeostasis, being involved in a range of key cellular and physiological processes. However, the mechanisms that drive functional activation of the transporters are largely unknown. In the present study, we investigated the function of a putative grapevine CAX-interacting protein, VvCXIP4, by testing its ability to activate VvCAX3, previously characterized as a tonoplast-localized Ca2+/H+ exchanger. VvCAX3 contains an autoinhibitory domain that drives inactivation of the transporter and thus, is incapable of suppressing the Ca2+-hypersensitive phenotype of the S. cerevisiae mutant K667. In this study, the co-expression of VvCXIP4 and VvCAX3 in this strain efficiently rescued its growth defect at high Ca2+ levels. Flow cytometry experiments showed that yeast harboring both proteins effectively accumulated higher Ca2+ levels than cells expressing each of the proteins separately. Bimolecular fluorescence complementation (BiFC) assays allowed visualization of the direct interaction between the proteins in tobacco plants and in yeast, and also showed the self-interaction of VvCAX3 but not of VvCXIP4. Subcellular localization studies showed that, despite being primarily localized to the nucleus, VvCXIP4 is able to move to other cell compartments upon a Ca2+ stimulus, becoming prone to interaction with the tonoplast-localized VvCAX3. qPCR analysis showed that both genes are more expressed in grapevine stems and leaves, followed by the roots, and that the steady-state transcript levels were higher in the pulp than in the skin of grape berries. Also, both VvCXIP4 and VvCAX3 were upregulated by Ca2+ and Na+, indicating they share common regulatory mechanisms. However, VvCXIP4 was also upregulated by Li+, Cu2+ and Mn2+, and its expression increased steadily throughout grape berry development, contrary to VvCAX3, suggesting additional physiological roles for VvCXIP4, including the regulation of VvCAXs not yet functionally characterized. The main novelty of the present study was the demonstration of physical interaction between CXIP and CAX proteins from a woody plant model by BiFC assays, demonstrating the intracellular mobilization of CXIPs in response to Ca2+.

[CAX3 Gene is Involved in Flax Response to High Soil Acidity and Aluminum Exposure].

Understanding the molecular mechanisms of plant response to unfavorable conditions is necessary for the effective selection of tolerant genotypes. Earlier, using high-throughput transcriptome sequencing of flax plants after exposure to aluminum ions (Al^(3+)) and high soil acidity, we detected stress-induced alteration in the expression of several genes, including CAX3, which encodes Ca^(2+)/H^(+)-exchanger involved in calcium ion transport. Here we describe CAX3 mRNA levels in flax cultivars either tolerant (Hermes and TMP1919) or sensitive (Lira and Orshanskiy) to Al^(3+). Stress-induced increased expression of CAX3 was detected only in aluminum-tolerant flax cultivars. The product of CAX3 gene may participate in flax response to high soil acidity and high Al^(3+) concentration through Ca^(2+)-mediated intracellular regulation.

The grapevine VvCAX3 is a cation/H+ exchanger involved in vacuolar Ca2+ homeostasis.

MAIN CONCLUSION: The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca 2+ , Na + and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca2+ homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca2+, demonstrating a role in Ca2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na+, Li+, Mn2+ and Cu2+ in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca2+, and Na+ in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.

Sugarcane ScCAX4 is a Negative Regulator of Resistance to Pathogen Infection.

Calcium (Ca2+) is a second messenger in various physiological processes within plants. The significance of the Ca2+/H+ exchanger (CAX) has been established in facilitating Ca2+ transport in plants; however, disease resistance functions of the CAX gene remain elusive. In this study, we conducted sequence characterization and expression analysis for a sugarcane CAX gene, ScCAX4 (GenBank Accession Number: MW206380). In order to further investigate the disease resistance functions, this gene was then transiently overexpressed in Nicotiana benthamiana leaves, which were subsequently inoculated with Fusarium solani var. coeruleum. Results showed that ScCAX4 overexpression increased the susceptibility of N. benthamiana to pathogen infection by regulating the expression of genes related to salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways, suggesting its negative role in disease resistance. Furthermore, we genetically transformed the ScCAX4 gene into N. benthamiana and obtained three positive T2 generation lines. Interestingly, the symptomatology of transgenic plants was consistent with that of transient overexpression after pathogen inoculation. Notably, the JA content in transgenic overexpression lines was significantly higher than that in the wild-type. RNA-seq revealed that ScCAX4 could mediate multiple signaling pathways, and the JA signaling pathway played a key role in modulating disease resistance. Finally, a regulatory model was depicted for the increased susceptibility to pathogen infection conferred by the ScCAX4 gene. This study provides genetic resources for sugarcane molecular breeding and the research direction for plant CAX genes.

Ectopic overexpression of Eleusine coracana CAX3 confers tolerance to metal and ion stress in yeast and Arabidopsis.

Ionic and metal toxicity in plants is still a global problem for the environment, agricultural productivity and ultimately poses human health threats when these metal ions accumulate in edible organs of plants. Metal and ion transport from cytosol to the vacuole is considered an important component of metal and ion tolerance and a plant's potential utility in phytoremediation. Finger millet (Eleusine coracana) is an orphan crop but has prominent nutritional value in comparison to other cereals. Previous transcriptomic studies suggested that one of the calcium/proton exchanger (EcCAX3) is strongly upregulated during different developmental stages of spikes development in plant. This finding led us to speculate that high calcium accumulation in the grain might be because of CAX3 function. Moreover, phylogenetic analysis shows that EcCAX3 is more closely related to foxtail millet, sorghum and rice CAX3 protein. To decipher the functional role of EcCAX3, we have adopted complementation of yeast triple mutant K677 (Δpmc1Δvcx1Δcnb1), which has defective calcium transport machinery. Furthermore, metal tolerance assay shows that EcCAX3 expression conferred tolerance to different metal stresses in yeast. The gain-of-function study suggests that EcCAX3 overexpressing Arabidopsis plants shows better tolerance to higher concentration of different metal ions as compared to wild type Col-0 plants. EcCAX3-overexpression transgenic lines exhibits abundance of metal transporters and cation exchanger transporter transcripts under metal stress conditions. Furthermore, EcCAX3-overexpression lines have higher accumulation of macro- and micro-elements under different metal stress. Overall, this finding highlights the functional role of EcCAX3 in the regulation of metal and ion homeostasis and this could be potentially utilized to engineer metal fortification and generation of stress tolerant crops in near future.

CAX control: multiple roles of vacuolar cation/H+ exchangers in metal tolerance, mineral nutrition and environmental signalling.

Plant vacuolar transporters, particularly CAX (Cation/H+ Exchangers) responsible for Ca2+/H+ exchange on the vacuole tonoplast, play a central role in governing cellular pH, ion balance, nutrient storage, metal accumulation, and stress responses. Furthermore, CAX variants have been employed to enhance the calcium content of crops, contributing to biofortification efforts. Recent research has uncovered the broader significance of these transporters in plant signal transduction and element partitioning. The use of genetically encoded Ca2+ sensors has begun to highlight the crucial role of CAX isoforms in generating cytosolic Ca2+ signals, underscoring their function as pivotal hubs in diverse environmental and developmental signalling networks. Interestingly, it has been observed that the loss of CAX function can be advantageous in specific stress conditions, both for biotic and abiotic stressors. Determining the optimal timing and approach for modulating the expression of CAX is a critical concern. In the future, strategically manipulating the temporal loss of CAX function in agriculturally important crops holds promise to bolster plant immunity, enhance cold tolerance, and fortify resilience against one of agriculture's most significant challenges, namely flooding.

The apple Ca2+/H+ exchanger MdCAX2L-2 functions positively in modulation of Ba2+ tolerance.

Calcium is an essential element for plant growth and development, and it plays an important role in the responses of plants to abiotic stress. High concentrations of heavy metal ions in soil significantly affect the yield and quality of crops and pose human health threats when these ions accumulate in edible organs. The Ca2+/H+ exchanger (CAX) family is a class of transporters that mediate the transmembrane transport of both Ca2+ and metal ions, and they are widely involved in regulating plant growth and development and stress responses. Here, we cloned an AtCAX2 ortholog, MdCAX2L-2, from apple. It is constitutively expressed in various apple tissues and significantly induced by Ca2+ and Ba2+ treatments. The MdCAX2L-2 protein is located in the vacuolar membrane in both plant and yeast cells. Overexpression of MdCAX2L-2 enhanced the tolerance of the yeast mutant K667 to high concentrations of Ca2+ and Ba2+. In addition, the role of MdCAX2L-2 in modulating Ba2+ tolerance was identified using MdCAX2L-2-overexpressing transgenic Arabidopsis plants and apple calli. Comparison of growth phenotypes and stress-related physiological indexes under BaCl2 treatment indicated that MdCAX2L-2 could enhance the Ba2+ tolerance of plants by promoting Ba2+ compartmentalization into the vacuoles and eliminating excess ROS. Our results provide insights that will aid future studies examining the function of CAX proteins in regulating stress tolerance in fruit crops, as well as their underlying mechanisms.

Redefining the N-Terminal Regulatory Region of the Ca2+/H+ Antiporter CAX1 in Tomato.

Calcium (Ca2+) is an essential plant nutrient, and Ca2+/H+ exchangers (CAXs) regulate Ca2+ partitioning between subcellular compartments. AtCAX1 activity is inhibited by its N-terminal regulatory region (NRR), which was initially defined as the sequence between the first two methionines. However, the accuracy of this NRR definition and the NRR regulatory mechanism remain unclear. Here, using tomato SlCAX1 as a model, we redefined the NRR of CAXs and demonstrated that our new definition is also applicable to Arabidopsis AtCAX1 and AtCAX3. The N-terminal-truncated SlCAX1 (SlCAX1Δ39) but not the full-length SlCAX1 was active in yeast, similar to Arabidopsis AtCAX1. Characterization of slcax1 mutants generated by CRISPR-Cas9 confirmed the calcium transport ability of SlCAX1. Sequence alignment between SlCAX1, AtCAX1, AtCAX3, and the Bacillus subtilis Ca2+/H+ antiporter protein YfkE revealed that SlCAX1 does not have the 2nd methionine and YfkE does not have any amino acid residues in front of the first transmembrane domain. Truncating the amino acid residues up to the first transmembrane of SlCAX1 (SlCAX1Δ66) further increased its activity. The same truncation had a similar effect on Arabidopsis AtCAX1 and AtCAX3. Expression of full-length SlCAX1 and SlCAX1Δ66 in tomato plants confirmed the results. Our results suggest that SlCAX1 is critical for Ca2+ homeostasis and all the amino acid residues in front of the first transmembrane domain inhibit the activity of CAXs. Our redefinition of the NRR will facilitate fine-tuning of Ca2+ partitioning to reduce the incidence of Ca2+-related physiological disorders in crops.

Genome-wide analysis and expression profiling of Cation/H+ exchanger (CAX) family genes reveal likely functions in cadmium stress responses in poplar.

Cadmium, a toxic heavy metal, seriously affects human health and ecological security. The cation/H+ exchanger (CAX) family is a unique metal transporter that plays a crucial role in Cd acquisition, transfer, and remission in plants. Although there are many studies related to the genome-wide analysis of Populus trichocarpa, little research has been done on the CAX family genes, especially concerning Cd stress. In this study, genome-wide analysis of the Populus CAX family identified seven stress-related CAX genes. The evolutionary tree indicated that the CaCA family genes were grouped into four clusters. Moreover, seven pairs of genes were derived by segmental duplication in poplars. Cis-acting element analysis identified numerous stress-related elements in the promoters of diverse PtrCAXs. Furthermore, some PtrCAXs were up-regulated by drought, beetle, and mechanical damage, indicating their possible function in regulating stress response. Under cadmium stress, all CAX genes in the roots were up-regulated. Our findings suggest that plants may regulate their response to Cd stress through the TF-CAXs module. Comprehensively investigating the CAX family provides a scientific basis for the phytoremediation of heavy metal pollution by Populus.

The Archaeal Na+/Ca2+ Exchanger (NCX_Mj) as a Model of Ion Transport for the Superfamily of Ca2+/CA Antiporters.

The superfamily of Calcium/Cation (Ca2+/CA) antiporters extrude Ca2+ from the cytosol or subcellular compartments in exchange with Na+, K+, H+, Li+, or Mg2+ and thereby provide a key mechanism for Ca2+ signaling and ion homeostasis in biological systems ranging from bacteria to humans. The structure-dynamic determinants of ion selectivity and transport rates remain unclear, although this is of primary physiological significance. Despite wide variances in the ion selectivity and transport rates, the Ca2+/CA proteins share structural motifs, although it remains unclear how the ion recognition/binding is coupled to the ion translocation events. Here, the archaeal Na+/Ca2+ exchanger (NCX_Mj) is considered as a structure-based model that can help to resolve the ion transport mechanisms by using X-ray, HDX-MS, ATR-FTIR, and computational approaches in conjunction with functional analyses of mutants. Accumulating data reveal that the local backbone dynamics at ion-coordinating residues is characteristically constrained in apo NCX_Mj, which may predefine the affinity and stability of ion-bound species in the ground and transition states. The 3Na+ or 1Ca2+ binding to respective sites of NCX_Mj rigidify the backbone dynamics at specific segments, where the ion-dependent compression of the ion-permeating four-helix bundle (TM2, TM3, TM7, and TM8) induces the sliding of the two-helix cluster (TM1/TM6) on the protein surface to switch the OF (outward-facing) and IF (inward-facing) conformations. Taking into account the common structural elements shared by Ca2+/CAs, NCX_Mj may serve as a model for studying the structure-dynamic and functional determinants of ion-coupled alternating access, transport catalysis, and ion selectivity in Ca2+/CA proteins.

Melatonin antagonizes ABA action to promote seed germination by regulating Ca2+ efflux and H2O2 accumulation.

Seed germination is a vital stage in the plant life-cycle that greatly contributes to plant establishment. Melatonin has been shown to promote seed germination under various environmental stresses; however, the mechanism remains largely underexplored. Here, we reported that melatonin antagonized abscisic acid (ABA) to promote seed germination by regulating ABA and gibberellic acid (GA3) balance. Transcriptomic analysis revealed that such a role of melatonin was associated with Ca2+ and redox signaling. Melatonin pretreatment induced Ca2+ efflux accompanied by an up-regulation of vacuolar H+/Ca2+ antiporter 3 (CAX3). AtCAX3 deletion in Arabidopsis exhibited reduced Ca2+ efflux. Inhibition of Ca2+ efflux in the seeds of melon and Arabidopsis mutant AtCAX3 compromised melatonin-induced germination under ABA stress. Melatonin increased H2O2 accumulation, and H2O2 pretreatment decreased ABA/GA3 ratio and promoted seed germination under ABA stress. However, complete inhibition of H2O2 accumulation abolished melatonin-induced ABA and GA3 balance and seed germination. Our study reveals a novel regulatory mechanism in which melatonin counteracts ABA to induce seed germination that essentially involves CAX3-mediated Ca2+ efflux and H2O2 accumulation, which, in turn, regulate ABA and GA3 balance by promoting ABA catabolism and/or GA3 biosynthesis.

The specificity of cyclists' aggression; examination of the cycling anger expression inventory across different recipient road user groups.

While cyclist aggression is relatively rare, it has been associated with increased crash risk. Previous measures of cyclist aggression have not taken into consideration who the recipient of the aggression may be; this is likely to differ across road user types. The aim of this study was to understand if cyclists' aggression differs according to whether the recipient is a driver, pedestrian or another cyclist. To examine this, 1206 cyclists (males = 75 %; age range 18-80 years; M = 47.35 ± 11.81) completed the Cycling Anger Expression Inventory (CAX; Møller and Haustein, 2017) three times; once for each road user type. Respondents also provided information regarding their cycling anger tendencies. Open text responses regarding sources of, and responses to, anger were also sought. The measurement invariance of the three CAX models was examined to determine whether the items were interpreted in a similar manner for interactions with each road user type and to compare latent factor means. The results showed that verbal aggression was not the same across road users. However, adaptive constructive ways of dealing with anger were similar across the three types of recipients. Comparison of latent means showed that cyclists reported higher expressions of anger toward drivers than cyclists or pedestrians. Qualitative analysis of the text responses suggest this is due to the perceived risk posed by drivers combined with positive attitudes expressed towards sharing infrastructure with pedestrians and other cyclists. Based on these findings it was concluded that: i) the CAX might best be used with reference to drivers, rather than "road users", and ii) while aggression in cyclists is rare, it is more common toward drivers than other road users.